In this project we determined antigenic relationships based on VP4 and VP7 neutralization specificities of various rotavirus strains derived from humans and animals. The elucidation of the neutralization specificities of rotavirus is important in order to achieve a more comprehensive understanding of rotavirus epidemiology and for formulation of an effective strategy for vaccination. By using various single gene substitution human x human or human x bovine rotavirus reassortants and hyperimmune guinea pig antiserum raised against each reassortant, we successfully characterized the VP4 neutralization specificity of representative human rotavirus strains (Wa, P, W161, DS-1, ST3, K8, 69M, Ro1845, PA169, and HAL1166) and established seven distinct VP4 (P) serotypes based on a <20-fold antibody difference criterion that is applied to classification of VP7 serotypes. The VP4 of vervet monkey rotavirus SA11 which has been used by many investigators as a prototype strain of group A rotaviruses was found to belong to serotype 5B which is represented by rhesus monkey rotavirus MMU18006 VP4. The US1205 strain (G serotype 9) which was originally isolated from a child with diarrhea in the U.S. was shown to carry VP4 serotype 2 (prototype strain ST3).